Single molecule-overlapping read analysis for minor variant mutation detection in pathogen samples

ABSTRACT

The present invention provides a method of detecting a heteroresistant population of a pathogen in a sample, the method comprising: a) providing a sample comprising a population of a pathogen; b) extracting nucleic acids from the sample; c) amplifying a target locus of the genome of the pathogen in the extracted nucleic acids, wherein the target locus comprises at least one minor variant associated with drug resistance in the pathogen; d) consecutively sequencing both overlapping nucleic acid strands from a single DNA molecule amplified from the target locus on a Next Generation Sequencing (NGS) platform; e) applying an alignment algorithm to sequencing data from the overlapping nucleic acid strands; and f) performing an analysis of the aligned sequencing data to detect the at least one minor variant and heteroresistant population of the pathogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/912,918, filed on Feb. 18, 2016 (published as US2016201115),which is the U.S. National Stage of International Patent Application No.PCT/US2014/052745, filed on Aug. 26, 2014, which claims the benefit ofU.S. Provisional Application No. 61/870,220 filed on Aug. 26, 2013, thecontents of each of which are hereby incorporated by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI082229 awardedby the National Institutes for Health. The government has certain rightsin this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide sequence listing submitted concurrently herewith andidentified as follows: One 1,761 byte ASCII (text) file named“91482_137_Sequence_Listing.txt” created on Aug. 26, 2014.

TECHNICAL FIELD

The present invention relates to diagnostic methods for the detectionand treatment of heteroresistant populations of pathogens.

BACKGROUND

Heteroresistance, the simultaneous occurrence of drug resistantsubpopulations in an otherwise drug susceptible bacterial population ina patient sample, has created uncertainty in the treatment and diagnosisof tuberculosis (TB) and is thought to be an important driver ofmulti-drug resistance in Mycobacterium tuberculosis (Mtb) which is asignificant threat to global TB control given its broadeningdistribution and the emergence of what is being called “totallydrug-resistant TB” (TDR-TB). It has been well-documented thatheteroresistance creates difficulties in the interpretation of rapidmolecular drug resistance tests because it leads to ‘indeterminate’ testresults, but the clinical significance of heteroresistance is stillbeing evaluated. Previously undetectable levels of Mtb resistantsubpopulations within a larger population of susceptible pathogens(i.e., heteroresistance) may be present in as many as 20% of TBpatients, and might help explain some of the inconsistency observed inTB treatment outcomes. Previously, there was no accurate means ofquantifying heteroresistance dynamics or even detecting the presence ofheteroresistance until the resistant sub-population had expanded to >1%of the pathogen population within a clinical sample. At that point,however, the patient's infection was determined to be resistant for thepurposes of treatment and a potential window of opportunity forpreventing resistance was closed.

The current gold standard method for Mtb drug susceptibility testing(DST) can be used to detect later stages of heteroresistance. Thisphenotypic test uses a culture-based, indirect proportion method whichrelies on the detection of growth of >1% of the inoculum on culturemedium containing a critical concentration of an anti-TB drug. It isestimated that for every 10⁷⁻⁸ Mtb bacilli there is at least onebacillus that has a genetic mutation that renders it naturally resistantto a particular drug. Suboptimal antibiotic treatment causes the few Mtbnaturally resistant mutants (by definition, a certain level ofheteroresistance) to gain a competitive advantage and subsequentlydominate the lesion. The 1% population component of resistant organismsthat is detectable by the DST method is well above naturally occurringMtb resistance mutation rates and, again, detections at this level aretypically too late to prevent treatment failure.

Current diagnostic methods are limited in their use and efficacy. Thethreshold of in vitro Mycobacteria Growth Indicator Tube (MGIT)heteroresistance detection is approximately 1% of the population, but isqualitative and takes weeks to complete. Current molecular tools thatdetect resistance-conferring mutations in Mtb coding genes and promoterregions which also have the potential to rapidly detectheteroresistance, include DNA sequencing (Sanger and pyrosequencing) andallele specific PCR analysis, but these methods are not sensitive orhave limited gene coverage. Sanger sequencing and pyrosequencing eachhave significant technical limitations for analyzing mixed populations.Sanger sequencing has a well-established detection threshold of ˜25%minor component in a mixed sample and relies on subjective visualevaluation of the electropherogram for “quantification”. Pyrosequencing(e.g., Qiagen Pyromark) is capable of true quantitative sequencing, buthas a reported quantification threshold of detection equal to 2.5%-5% ofan Mtb population and has limited sequencing depth capability. ThePCR-hybridization approach of the line-probe assay has a describedsensitivity of detection of a minor resistant variant detection at 5%.Detection of heteroresistance, even at the 1% level provided byMGIT-DST, is insufficient and likely too late to prevent treatmentfailure. A quantitative and more sensitive method is highly desirable.

Mtb heteroresistance is not a rare phenomenon, occurring in 9-30% of Mtbpopulations studied, and has been identified in Mtb populations withphenotypic resistance to first line-drugs (INH, RIF, ETH, and STR) andsecond-line fluoroquinolones (ofloxacin-OFX) and injectables (AMK). Itis highly likely that drug resistant organisms are present in most TBlesions, even as very minor population components, given the highbacilli loads that are typically found in patients.

There is also a critical need to detect minor resistance variantpopulations in clinical samples early in therapy to allow forcustomizable patient treatment and to track variant populations'progress through time. The present invention addresses this need byproviding methods to detect heteroresistance in TB patient samples atlow resistance levels using multiple target loci amplification andsequencing with clinically relevant next generation technology. Thistranslates to a significant increase in the level of detectionsensitivity as compared to other methods and an enhanced ability totreat patients with effective therapies against the resistant pathogenpopulations.

SUMMARY

In some embodiments, the present invention is directed to a method ofdetecting a heteroresistant population of a pathogen in a sample, themethod comprising: a) providing a sample comprising a population of apathogen; b) extracting nucleic acids from the sample; c) amplifying atarget locus of the genome of the pathogen in the extracted nucleicacids, wherein the target locus comprises at least one minor variantassociated with drug resistance in the pathogen; d) consecutivelysequencing both overlapping nucleic acid strands from a single DNAmolecule amplified from the target locus on a Next Generation Sequencing(NGS) platform; e) applying an alignment algorithm to sequencing datafrom the overlapping nucleic acid strands; and f) performing an analysisof the aligned sequencing data to detect the at least one minor variantand heteroresistant population of the pathogen. In certain embodiments,both overlapping nucleic acid strands refers to the coding strand andthe noncoding strand of a gene (e.g., a resistance gene such as, but notlimited torrs, katG, inhA, and gyrA).

In some implementations, the analysis of the aligned sequencing data isa minor variant analysis. In certain aspects, the minor variant analysisis a haplotype variant analysis. The target locus may be amplified witha high fidelity polymerase such as KAPA HiFi™ DNA polymerase or Q5® HIFIDNA polymerase.

In some aspects, each of the overlapping nucleic acid strands consistsof less than about 500 nucleotides, less than about 450 nucleotides,less than about 400 nucleotides, less than about 350 nucleotides, lessthan about 300 nucleotides, less than about 250 nucleotides, less thanabout 200 nucleotides, less than about 150 nucleotides, less than about100 nucleotides, or less than about 50 nucleotides.

In other aspects, the alignment algorithm is optimized for shortnucleotide space reads of less than about 500 nucleotides, less thanabout 450 nucleotides, less than about 400 nucleotides, less than about350 nucleotides, less than about 300 nucleotides, less than about 250nucleotides, less than about 200 nucleotides, less than about 150nucleotides, less than about 100 nucleotides, or less than about 50nucleotides. The alignment algorithm may be Novoalign. Various NGSplatforms may be used with the present invention including the IluminaMiSeq platform.

In some aspects, the minor variant analysis is performed with abioinformatics script that requires a user to input genomic regions ofinterest and generates a report with single molecule-overlapping readinformation used to identify the minor variant.

In yet other aspects, the methods of the present invention furthercomprise using a highly homogenous synthetic plasmid standard toidentify actual sequence error rate variance between target loci andsequencing runs.

In some embodiments, the minor variant is selected from the groupconsisting of a single nucleotide polymorphism (SNP), an insertion, anda deletion.

In other embodiments, the pathogen is Mycobacterium tuberculosis. Inthese embodiments, the at least one minor variant may be located withina genomic sequence selected from the group consisting of katG, inhApromoter, rpoB, gyrA, rrs, eis promoter, and combinations thereof.

In certain aspects, the heteroresistant population is resistant toisoniazid (INH), rifampin (RIF), moxifloxacin (MOX), amikacin (AMK),kanamycin (KAN) and/or capreomycin (CAP).

In some embodiments, the present invention is directed to methods oftreating a subject in need thereof with a therapeutic agent to aheteroresistant population of M. tuberculosis, wherein the therapeuticagent is selected from the group consisting of PA-824, OPC-67683, SQ109,TMC207, NAS-21, NAS-91, thioridazine, chlorpromazine, and a1,3-benzothiazin-4-one, and combinations thereof. In certain aspects,the treatment is preceded by the detection of one or moreheteroresistant population of M. tuberculosis in a sample from thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative and exemplary embodiments of the invention are shown in thedrawings in which:

FIG. 1 depicts a model of heteroresistant dynamics of Mtb in anon-compliant patient.

FIG. 2 depicts SMOR analysis of reads for inhA and gyrA.

FIG. 3 depicts mixed read alignment to gyrA from overlapping readanalysis with six different sequences shown (SEQ ID NOs: 1-6). A mixtureof resistant (1%) and susceptible (99%) alleles was sequencedto >224,000× coverage and the alignment of 18 reads (9 paired sets) isshown to illustrate the detection of minor variants. Random single SNPsoccurring on one strand but not their complement are distinguished fromreal variants (two examples) as sequencing errors.

FIG. 4 depicts a sample analysis pipeline with a SMOR heteroresistancedetection assay.

Elements and facts in the figures are illustrated for simplicity andhave not necessarily been rendered according to any particular sequenceor embodiment.

DETAILED DESCRIPTION

Aspects and applications of the invention presented herein are describedbelow in the drawings and detailed description of the invention. Unlessspecifically noted, it is intended that the words and phrases in thespecification and the claims be given their plain, ordinary, andaccustomed meaning to those of ordinary skill in the applicable arts.

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the invention. It will beunderstood, however, by those skilled in the relevant arts, that thepresent invention may be practiced without these specific details. Thefull scope of the inventions is not limited to the specific examplesthat are described below.

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one”.

As used herein, a “sample,” such as a biological sample that includesnucleic acid molecules, is a sample obtained from a subject. As usedherein, biological samples include all clinical samples including, butnot limited to, cells, tissues, and bodily fluids, such as: blood;derivatives and fractions of blood, such as serum; extracted galls;biopsied or surgically removed tissue, including tissues that are, forexample, unfixed, frozen, fixed in formalin and/or embedded in paraffin;tears; milk; skin scrapes; surface washings; urine; sputum;cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates. In aparticular example, a sample includes blood obtained from a humansubject, such as whole blood or serum. In another particular example, asample includes buccal cells, for example collected using a swab or byan oral rinse.

In some embodiments, the present invention is directed to anext-generation sequencing analysis methodology to detect minorproportions of a sample that contain mutations associated with importantphenotypes, including antibacterial and antiviral resistance. Thisanalysis decreases the sequencing error rate so that extremely lowlevels of true minor components (e.g., SNP loci) can be detected.

The incidence of drug-resistant (DR) tuberculosis (TB) continues toincrease worldwide. Undetected heteroresistance, the presence of DR andsusceptible genotypes in bacterial populations involved in infection, attreatment initiation may play a role in the expansion of DR strains andtreatment failure. In Mycobacterium tuberculosis, current minor DRcomponent detection levels are limited to ˜1%, using phenotypic drugsusceptibility testing, which requires 15-30 days or even longer tocomplete. By that point during an infection, it is likely too late toprevent DR-TB and treatment failure.

In some aspects, the present invention relates to a method of detectingresistant Mtb sub-populations consisting of 0.1% or less of the totalMtb population in under a week. Detection of minor components in complexbiological mixtures has radically advanced with the emergence ofnext-generation sequencing. Low-level detection from sequence data,however, is not trivial, primarily due to the error rates in sequencing.The error associated with the respective sequencing platform, as well asthe GC content of the organism, sets the limit of discerning actualminor component from error. However, the use of “singlemolecule-overlapping reads” (SMOR) analysis for determination of actualmutation ratios in target loci (e.g., antibiotic resistance genes) leadsto an increase in heteroresistance detection sensitivity and lower errorbias.

The use of overlapping reads allows for effective coverage of each locuson both strands of an individual sequenced DNA molecule, which in turnallows for an independent confirmation of the specific nucleotide atthat single locus. The product rule of probability applies, such that ifone locus on a single molecule is read two times, it has the lower limitof detection of the probability of one error occurring squared. In someembodiments, the Illumina Miseq platform is used to sequence ampliconsfrom several different in vitro mixtures of DR and susceptible Mtbstrains to validate the use of SMOR for identifying heteroresistance.The calculated average of combined amplification and sequencing errorrate for Mtb (a high GC organism) is 0.51% per position across theamplicons tested. When employing SMOR, the theoretical limit ofdetection of a minor component is 2.6×10⁻⁶, readily allowing fordetection of minor components below 0.51%.

The Inventors have been able to detect a 0.3% artificial mixture of SNPalleles in the inhA promoter at a frequency of 3.07×10⁻³, which was atleast two orders of magnitude more frequent than identifiable sequenceerrors. The use of SMOR allows for researchers and clinicians to followthe evolution of heteroresistance, determine its clinical relevance anddevelop appropriate treatment strategies to suppress minor componentresistant sub-populations before they become clinically significant.

In Mycobacteria tuberculosis (Mtb) there are characterized SNPs thatconfer resistance to several different antibiotics. By using overlappingreads on these targeted regions we can characterize heteroresistance inclinical samples down to a level that has not been previously achieved.Overlapping reads have been used in next generation sequencing toimprove whole genome examination but they have not been used to addconfidence in antibiotic resistance population evaluation.

With the invention, clinicians are able to track patient treatment in amore timely fashion and alter the course of treatment whenheteroresistance is detected within a week versus a month or more as iscommon with current technology. This analysis can also be useful toresearchers wanting to characterize population structure within a singlesample of bacteria.

In one embodiment, the invention provides a diagnostic assay for thedetection of heteroresistance in Mycobacterium tuberculosis in clinicalsamples. Modifications of this assay can be used for low level minormutation resistance in other organisms, as we have also demonstratedwith influenza.

In some embodiments, the limit of detection for the minor variant isless than about 1.0%, less than about 0.9%, less than about 0.8%, lessthan about 0.7%, less than about 0.6%, less than about 0.5%, less thanabout 0.4%, less than about 0.3%, less than about 0.2%, less than about0.1%, less than about 0.09%, less than about 0.08%, less than about0.07%, less than about 0.06%, less than about 0.05%, less than about0.04%, less than about 0.03%, less than about 0.02%, or less than about0.01% of the heteroresistant population.

In other embodiments, each of the overlapping nucleic acid strands to besequenced with the disclosed method consists of less than about 500nucleotides, less than about 450 nucleotides, less than about 400nucleotides, less than about 350 nucleotides, less than about 300nucleotides, less than about 250 nucleotides, less than about 200nucleotides, less than about 150 nucleotides, less than about 100nucleotides, or less than about 50 nucleotides.

In yet other embodiments, the disclosed method further comprisesadministering a therapeutic agent to a heteroresistant population of M.tuberculosis. Exemplary therapeutic agents are found in Da Silva et al.(2011) J. Antimicrob. Chemother. 66:1417.

The minor variant detected in the heteroresistant population of M.tuberculosis may be an SNP, an insertion, or a deletion. Non-limitingexamples of genetic mutations associated with drug resistance in M.tuberculosis are found in Georghiou et al. (2012) PLoS ONE 7(3):e33275.

Mathematical models of within-host Mtb population dynamics havepredicted that heteroresistance can cause the emergence of MDR-TB priorto treatment initiation, and this emergence may occur 1,000-10,000 timesmore frequently. Studies of within-host dynamics of Mtb growth duringtreatment of have also indicated that resistant subpopulations caneasily dominate a lesion over time in both treatment compliant andnon-compliant patients (FIG. 1). The presence of resistance conferringmutations, even as minor components of an infecting population of Mtb,likely leads to selection of resistant strains, in the presence of thecorresponding drug, and subsequent treatment failure. Minor resistantpopulations, however, are typically missed through standard analysis ofisolates because the dominant organism phenotype masks any minorcomponent variants. In certain aspects, the present invention addressesthis problem by providing effective methods to detect and quantify minorresistant populations.

In some embodiments, the present invention is directed to the detectionand analysis of heteroresistance in tuberculosis infections. An assay isprovided that is able to accurately detect heteroresistance in Mtb andquantify the presence and proportion of all resistant allele minorcomponents down to less than 0.1% using clinically relevant table-topnext generation sequencing (NGS) technology and advanced bioinformaticalgorithms. This approach provides a rapid, highly sensitive andspecific method for detecting and monitoring the potential clinicalrelevance of heteroresistance in serial clinical samples from TBpatients, which is not achievable by any other existing technology.Additionally, the NGS technology used in the assay can be used for deepsequencing of multiple targeted areas simultaneously, which allows forthe detection of extremely rare minor components in a population at alltargeted locations at once. This multiplexing approach is ideal fordeveloping a practical, efficient and rapid analysis of heteroresistancedirectly from patient sputum, which has significant advantages overexisting technologies.

While deep-sequencing seems to be an obvious solution, it is notsufficient, in and of itself. NGS minor variant detection is nottrivial; primarily due to the error rates associated with the sequencingplatform (e.g. Illumina MiSeq platform has a standard rating of 75% ofbases having a 0.1% error). This rate sets a theoretical limit ofdiscerning a rare variant from error but recent advances in technologyand bioinformatics allow for minor variant detection at significantlylower levels than expected error rate. An advantage resulting from theapproach of the present invention is the ability to accurately detectminor components below the sequencing error by using a “Single-MoleculeOverlapping Read” (SMOR) analysis.

In certain aspects, the present invention relates to an approach toapplying cutting-edge genomic science and technology to the ongoingclinical and public health problem of multi-drug resistant tuberculosis.In one embodiment, an optimized heteroresistance assay is used to detectknown mutations associated with six anti-TB drugs, followed by anevaluation of heteroresistance in serial samples from a patientpopulation.

In certain aspects, the population of bacteria comprises one or morebacteria selected from the group consisting of Actinomedurae,Actinomyces israelii, Bacillus anthraces, Bacillus cereus, Clostridiumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridiumtetani, Corynebacterium, Enterococcus faecalis, Listeria monocytogenes,Nocardia, Propionibacterium acnes, Staphylococcus aureus, Staphylococcusepiderm, Streptococcus mutans, Streptococcus pneumonia, Afipia felis,Bacteriodes, Bartonella bacilliformis, Bortadella pertussis, Borreliaburgdorferi, Borrelia recurrentis, Brucella, Calymmatobacteriumgranulomatis, Campylobacter, Escherichia coli, Francisella tularensis,Gardnerella vaginalis, Haemophilius aegyptius, Haemophilius ducreyi,Haemophilius influenziae, Heliobacter pylori, Legionella pneumophila,Leptospira interrogans, Neisseria meningitidia, Porphyromonasgingivalis, Providencia sturti, Pseudomonas aeruginosa, Salmonellaenteridis, Salmonella typhi, Serratia marcescens, Shigella boydii,Streptobacillus moniliformis, Streptococcus pyogenes, Treponemapallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis,Bartonella henselae, Chlamydia psittaci, Chlamydia trachomatis, Coxiellaburnetii, Mycoplasma pneumoniae, Rickettsia akari, Rickettsiaprowazekii, Rickettsia rickettsia, Rickettsia tsutsugamushi, Rickettsiatyphi, Ureaplasma urealyticum, Diplococcus pneumoniae, Ehrlichiachafensis, Enterococcus faecium, Meningococci, Burkholderia mallei,Burkholderia pseudomallei, Ricinus communis, and Cryptosporidium parvum.

In some embodiments, the present invention further comprisesadministering to the subject a regime of antibiotics to effectivelycontrol the population of pathogen based on the presence or absence ofantibiotic resistance markers in the pathogen.

In certain aspects, the present invention is used to detect and monitorantibiotic resistance in a subject infected with a population ofbacteria. Antibiotic resistance can be determined by the presence orabsence of one or more antibiotic resistance genes or markers in thepopulation. Non-limiting examples of such antibiotic resistance genesinclude bla_(tem), bla_(shv), bla_(rob), bla_(oxa), blaZ, aadB, aacC1,aacC2, aacC3, aac6′-IIa, aacA4, aad(6′), vanA, vanB, vanC, msrA, sarA,aac(6′) aph(2″), vat, vga, ermA, ermB, ermC, mecA, int, sul, mecA,aac2ia, aac2ib, aac2ic, aac2id, aac2i, aac3ia, aac3iia, aac3iib,aac3iii, aac3iv, aac3ix, aac3vi, aac3viii, aac3vii, aac3x, aac6i,aac6ia, aac6ib, aac6ic, aac6ie, aac6if, aac6ig, aac6iia, aac6iib, aad9,aad9ib, aadd, acra, acrb, adea, adeb, adec, amra, amrb, ant2ia, ant2ib,ant3ia, ant4iia, ant6ia, aph33ia, aph33ib, aph3ia, aph3ib, aph3ic,aph3iiia, aph3iva, aph3va, aph3vb, aph3via, aph3viia, aph4ib, aph6ia,aph6ib, aph6ic, aph6id, arna, baca, bcra, bcrc, bl1_acc, bl1_ampc,bl1_asba, bl1_ceps, bl1_cmy2, bl1_ec, bl1_fox, bl1_mox, bl1_och,bl1_pao, bl1_pse, bl1_sm, bl2a_1, bl2a_exo, bl2a_iii2, bl2a_iii,bl2a_kcc, bl2a_nps, bl2a_okp, bl2a_pc, bl2be_ctxm, bl2be_oxy1,bl2be_per, bl2be_shv2, bl2b_rob, bl2b_tem1, bl2b_tem2, bl2b_tem,bl2b_tle, bl2b_ula, bl2c_bro, bl2c_pse1, bl2c_pse3, bl2d_ler1,bl2d_moxa, bl2d_oxa10, bl2d_oxa1, bl2d_oxa2, bl2d_oxa5, bl2d_oxa9,bl2d_r39, bl2e_cbla, bl2e_cepa, bl2e_cfxa, bl2e_fpm, bl2e_y56,bl2f_nmca, bl2f_sme1, bl2_ges, bl2_kpc, bl2_len, bl2_veb, bl3_ccra,bl3_cit, bl3_cpha, bl3_gim, bl3_imp, bl3_1, bl3_shw, bl3_sim, bl3_vim,ble, blt, bmr, cara, cata10, cata11, cata12, cata13, cata14, cata15,cata16, cata1, cata2, cata3, cata4, cata5, cata6, cata7, cata8, cata9,catb1, catb2, catb3, catb4, catb5, ceoa, ceob, cml_e1, cml_e2, cml_e3,cml_e4, cml_e5, cml_e6, cml_e7, cml_e8, dfra10, dfra12, dfra13, dfra14,dfra15, dfra16, dfra17, dfra19, dfra1, dfra20, dfra21, dfra22, dfra23,dfra24, dfra25, dfra25, dfra25, dfra26, dfra5, dfra7, dfrb1, dfrb2,dfrb3, dfrb6, emea, emrd, emre, erea, ereb, erma, ermb, ermc, ermd,erme, ermf, ermg, ermh, ermn, ermo, ermq, ermr, erms, emrt, ermu, ermv,ermw, ermx, ermy, fosa, fosb, fosc, fosx, fusb, fush, ksga, lmra, lmrb,lnua, lnub, lsa, maca, macb, mdte, mdtf, mdtg, mdth, mdtk, mdtl, mdtm,mdtn, mdto, mdtp, meca, mecr1, mefa, mepa, mexa, mexb, mexc, mexd, mexe,mexf, mexh, mexi, mexw, mexx, mexy, mfpa, mpha, mphb, mphc, msra, norm,oleb, opcm, opra, oprd, oprj, oprm, oprn, otra, otrb, pbp1a, pbp1b,pbp2b, pbp2, pbp2x, pmra, qac, qaca, qacb, qnra, qnrb, qnrs, rosa, rosb,smea, smeb, smec, smed, smee, smef, srmb, sta, str, sul1, sul2, sul3,tcma, tcr3, tet30, tet31, tet32, tet33, tet34, tet36, tet37, tet38,tet39, tet40, teta, tetb, tetc, tetd, tete, tetg, teth, tetj, tetk,tetl, tetm, teto, tetpa, tetpb, tet, tetq, tets, tett, tetu, tetv, tetw,text, tety, tetz, tlrc, tmrb, tolc, tsnr, vana, vanb, vanc, vand, vane,vang, vanha, vanhb, vanhd, vanra, vanrb, vanrc, vanrd, valre, vanrg,vansa, vansb, vansc, vansd, vanse, vansg, vant, vante, vantg, vanug,vanwb, vanwg, vanxa, vanxb, vanxd, vanxyc, vanxye, vanxyg, vanya, vanyb,vanyd, vanyg, vanz, vata, vatb, vatc, vatd, vale, vgaa, vgab, vgba,vgbb, vph, ykkc, and ykkd (see the Antibiotic Resistance Genes Database(ARDB) available online).

In certain embodiments, the method of the present invention furthercomprises treating the subject with an antibiotic or regime ofantibiotics. Non-limiting examples of such antibiotics includeamoxillin, erythromycin, azithromycin, clarithromycin, gentamicin,tobramycin, ciprofloxaxin, norfloxacin, gatifloxacin, ofloxacin,levofloxacin, moxifloxacin, metronidazole, lomefloxacin, ciprofloxacin,natamycin, neomycin, polymyxin B, gentamycin, bacitracin, trovafloxacin,grepafloxacin, sulfacetamide, tetracycline, gramicidin, chloramphenicol,or gramicidin. amino glycosides (gentamicin, neomycin, kanamycin,tobramycin, framycetin, streptomycin, amikacin), ampicillin andamoxillin, sulphonamides (trimethoprim-sulfamethoazole), cephalosporins,groups of beta-lactams, chloramphenicols, lincosamides, macrolides,penicillin, group of quinolones, tetracyclins andnitrafuratoin/nitrofurazone, polymyxin B, mupirocin, vancomycin,antimicrobial agents containing one or more biguanide groups (forexample chlorhexidine or PHMB), silver complexes or silver salts,hydrogenperoxide and other oxidizing agents, quaternary ammoniumcompounds, agents delivering chlorine, and antimicrobial peptides.

In some embodiments, the nucleic acids from the sample is analyzed bySequencing by Synthesis (SBS) techniques. SBS techniques generallyinvolve the enzymatic extension of a nascent nucleic acid strand throughthe iterative addition of nucleotides against a template strand. Intraditional methods of SBS, a single nucleotide monomer may be providedto a target nucleotide in the presence of a polymerase in each delivery.However, in some of the methods described herein, more than one type ofnucleotide monomer can be provided to a target nucleic acid in thepresence of a polymerase in a delivery.

SBS can utilize nucleotide monomers that have a terminator moiety orthose that lack any terminator moieties. Methods utilizing nucleotidemonomers lacking terminators include, for example, pyrosequencing andsequencing using γ-phosphate-labeled nucleotides. In methods usingnucleotide monomers lacking terminators, the number of differentnucleotides added in each cycle can be dependent upon the templatesequence and the mode of nucleotide delivery. For SBS techniques thatutilize nucleotide monomers having a terminator moiety, the terminatorcan be effectively irreversible under the sequencing conditions used asis the case for traditional Sanger sequencing which utilizesdideoxynucleotides, or the terminator can be reversible as is the casefor sequencing methods developed by Solexa (now Illumina, Inc.). Inpreferred methods a terminator moiety can be reversibly terminating.

SBS techniques can utilize nucleotide monomers that have a label moietyor those that lack a label moiety. Accordingly, incorporation events canbe detected based on a characteristic of the label, such as fluorescenceof the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a byproduct of incorporation of thenucleotide, such as release of pyrophosphate; or the like. Inembodiments, where two or more different nucleotides are present in asequencing reagent, the different nucleotides can be distinguishablefrom each other, or alternatively, the two or more different labels canbe the indistinguishable under the detection techniques being used. Forexample, the different nucleotides present in a sequencing reagent canhave different labels and they can be distinguished using appropriateoptics as exemplified by the sequencing methods developed by Solexa (nowIllumina, Inc.). However, it is also possible to use the same label forthe two or more different nucleotides present in a sequencing reagent orto use detection optics that do not necessarily distinguish thedifferent labels. Thus, in a doublet sequencing reagent having a mixtureof A/C both the A and C can be labeled with the same fluorophore.Furthermore, when doublet delivery methods are used all of the differentnucleotide monomers can have the same label or different labels can beused, for example, to distinguish one mixture of different nucleotidemonomers from a second mixture of nucleotide monomers. For example,using the [First delivery nucleotide monomers]+[Second deliverynucleotide monomers] nomenclature set forth above and taking an exampleof A/C+(1/T), the A and C monomers can have the same first label and theG and T monomers can have the same second label, wherein the first labelis different from the second label. Alternatively, the first label canbe the same as the second label and incorporation events of the firstdelivery can be distinguished from incorporation events of the seconddelivery based on the temporal separation of cycles in an SBS protocol.Accordingly, a low resolution sequence representation obtained from suchmixtures will be degenerate for two pairs of nucleotides (T/G, which iscomplementary to A and C, respectively; and C/A which is complementaryto G/T, respectively).

Some embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891;6,258,568 and 6,274,320, the disclosures of which are incorporatedherein by reference in their entireties). In pyrosequencing, releasedPPi can be detected by being immediately converted to adenosinetriphosphate (ATP) by ATP sulfurylase, and the level of ATP generated isdetected via luciferase-produced photons.

In another example type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in U.S. Pat. Nos. 7,427,67, 7,414,1163 and 7,057,026, thedisclosures of which are incorporated herein by reference. This approachis being commercialized by Solexa (now Illumina Inc.), and is alsodescribed in WO 91/06678 and WO 07/123,744 (filed in the United StatesPatent and Trademark Office as U.S. Ser. No. 12/295,337), each of whichis incorporated herein by reference in their entireties. Theavailability of fluorescently-labeled terminators in which both thetermination can be reversed and the fluorescent label cleavedfacilitates efficient cyclic reversible termination (CRT) sequencing.Polymerases can also be co-engineered to efficiently incorporate andextend from these modified nucleotides.

In other embodiments, Ion Semiconductor Sequencing is utilized toanalyze the nucleic acids from the sample. Ion Semiconductor Sequencingis a method of DNA sequencing based on the detection of hydrogen ionsthat are released during DNA amplification. This is a method of“sequencing by synthesis,” during which a complementary strand is builtbased on the sequence of a template strand.

For example, a microwell containing a template DNA strand to besequenced can be flooded with a single species of deoxyribonucleotide(dNTP). If the introduced dNTP is complementary to the leading templatenucleotide it is incorporated into the growing complementary strand.This causes the release of a hydrogen ion that triggers a hypersensitiveion sensor, which indicates that a reaction has occurred. If homopolymerrepeats are present in the template sequence multiple dNTP moleculeswill be incorporated in a single cycle. This leads to a correspondingnumber of released hydrogens and a proportionally higher electronicsignal.

This technology differs from other sequencing technologies in that nomodified nucleotides or optics are used. Ion semiconductor sequencingmay also be referred to as ion torrent sequencing, proton-mediatedsequencing, silicon sequencing, or semiconductor sequencing. Ionsemiconductor sequencing was developed by Ion Torrent Systems Inc. andmay be performed using a bench top machine. Rusk, N. (2011). “Torrentsof Sequence,” Nat Meth 8(1): 44-44. Although it is not necessary tounderstand the mechanism of an invention, it is believed that hydrogenion release occurs during nucleic acid amplification because of theformation of a covalent bond and the release of pyrophosphate and acharged hydrogen ion. Ion semiconductor sequencing exploits these factsby determining if a hydrogen ion is released upon providing a singlespecies of dNTP to the reaction.

For example, microwells on a semiconductor chip that each contain onesingle-stranded template DNA molecule to be sequenced and one DNApolymerase can be sequentially flooded with unmodified A, C, G or TdNTP. Pennisi, E. (2010). “Semiconductors inspire new sequencingtechnologies” Science 327(5970): 1190; and Perkel, J., “Making contactwith sequencing's fourth generation” Biotechniques (2011). The hydrogenion that is released in the reaction changes the pH of the solution,which is detected by a hypersensitive ion sensor. The unattached dNTPmolecules are washed out before the next cycle when a different dNTPspecies is introduced.

Beneath the layer of microwells is an ion sensitive layer, below whichis a hypersensitive ISFET ion sensor. All layers are contained within aCMOS semiconductor chip, similar to that used in the electronicsindustry. Each released hydrogen ion triggers the ISFET ion sensor. Theseries of electrical pulses transmitted from the chip to a computer istranslated into a DNA sequence, with no intermediate signal conversionrequired. Each chip contains an array of microwells with correspondingISFET detectors. Because nucleotide incorporation events are measureddirectly by electronics, the use of labeled nucleotides and opticalmeasurements are avoided.

An example of a Ion Semiconductor Sequencing technique suitable for usein the methods of the provided disclosure is Ion Torrent sequencing(U.S. Patent Application Numbers 2009/0026082, 2009/0127589,2010/0035252, 2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617,2010/0300559), 2010/0300895, 2010/0301398, and 2010/0304982), thecontent of each of which is incorporated by reference herein in itsentirety. In Ion Torrent sequencing, DNA is sheared into fragments ofapproximately 300-800 base pairs, and the fragments are blunt ended.Oligonucleotide adaptors are then ligated to the ends of the fragments.The adaptors serve as primers for amplification and sequencing of thefragments. The fragments can be attached to a surface and are attachedat a resolution such that the fragments are individually resolvable.Addition of one or more nucleotides releases a proton (H+), which signaldetected and recorded in a sequencing instrument. The signal strength isproportional to the number of nucleotides incorporated. User guidesdescribe in detail the Ion Torrent protocol(s) that are suitable for usein methods of the invention, such as Life Technologies' literatureentitled “Ion Sequencing Kit for User Guide v. 2.0” for use with theirsequencing platform the Personal Genome Machine™ (PCG).

In some embodiments, as a part of the sample preparation process,“barcodes” may be associated with each sample. In this process, shortoligos are added to primers, where each different sample uses adifferent oligo in addition to a primer.

The term “library”, as used herein refers to a library of genome-derivedsequences. The library may also have sequences allowing amplification ofthe “library” by the polymerase chain reaction or other in vitroamplification methods well known to those skilled in the art. Thelibrary may also have sequences that are compatible with next-generationhigh throughput sequencers such as an ion semiconductor sequencingplatform.

In certain embodiments, the primers and barcodes are ligated to eachsample as part of the library generation process. Thus during theamplification process associated with generating the ion ampliconlibrary, the primer and the short oligo are also amplified. As theassociation of the barcode is done as part of the library preparationprocess, it is possible to use more than one library, and thus more thanone sample. Synthetic DNA barcodes may be included as part of theprimer, where a different synthetic DNA barcode may be used for eachlibrary. In some embodiments, different libraries may be mixed as theyare introduced to a flow cell, and the identity of each sample may bedetermined as part of the sequencing process. Sample separation methodscan be used in conjunction with sample identifiers. For example a chipcould have 4 separate channels and use 4 different barcodes to allow thesimultaneous running of 16 different samples.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLES Example 1 Mycobacterium Tuberculosis (Mtb) HeteroresistanceAssay Optimization and Validation

The goal of this study was to detect and quantify the presence ofresistant alleles associated with primary TB drug (INH, RIF, MOX, AMK,KAN, and CAP) resistance that occur in ≤0.1% of a mixed(resistant/susceptible) population of Mtb in clinical samples. Toaccomplish this goal the Inventors optimized and validated a prototypeMtb heteroresistance assay based on NGS technology, which can accuratelydetect resistant alleles (minor components) down to less than 0.1%, forall known SNPs associated with XDR-TB in six primary resistance genes.

The Inventors exploited the deep-sequencing capability of the IlluminaNGS platforms to detect extremely rare minor components in an Mtbpopulation. The deep-sequencing coverage capacity is dependent on thecomplexity of the genomic material being sequenced; in the case ofresistance in Mtb the Inventors focused on sequencing amplicons (˜300bp) that contain drug resistance target loci: SNPs previouslydemonstrated to predict XDR-TB phenotypic resistance with 90-98%sensitivity and ˜100% specificity. This short genomic length allows for10⁴×-10⁵× coverage for each individual amplicon, for all targetresistance gene regions, for up to 100 distinct clinical samples on asingle Illumina MiSeq run. However, as stated above, the lower limit ofdetection for each targeted loci is directly related to the sequencingerror rate, not the coverage. The Inventors advanced the ability todetect extremely rare SNP alleles below the error rate using highfidelity Taq polymerase, improved alignment algorithms, advancedbioinformatics, and minor variant analysis. The use of high fidelitythermostable polymerases reduces the impact of PCR-amplification erroron minor variant detection. The error rate of KAPA HiFi (2.8×10⁻⁷) is100× lower than Taq polymerase and 40× lower than polymerase blends suchas PLATINUM® Taq High Fidelity. Improvements in alignment algorithmshave allowed for more accurate assemblies and alignments of sequencedata, which result in the reduction of the overall error rate. Advancedbioinformatics tools, developed in house, better handle the extremesequence coverage (>100,000×), allowing for the detection of ultra-rarevariants.

The most significant advance in minor variant detection has come from“haplotype variant analysis”, which involves the detection andquantitation of multiple independent variant loci. In short, haplotypevariant analysis predicts that the lower limit of minor componentdetection decreases by the product of individual error rates for thenumber of target loci contained in an individual sequence read (Box 1).Therefore, if only one SNP loci is interrogated and that loci has aknown sequencing error rate of 10⁻³ (0.1%), then the theoretical lowerlimit of detection (LOD) is >10⁻³ and is greatly influenced by thesequence coverage. However, if two separate SNP loci are interrogated ona single read, each of which has a sequencing error of 10⁻³, then theirhaplotype combined error rate is the product of the individual errorrates, or 10⁻⁶; for the sake of a simplified probabilistic discussion(Box 1), a conservative LOD that is 10-fold higher or 10⁻⁵ (0.001%) isused. The Inventors used this product rule of probability characteristicof haplotype variant analysis to establish a deep-sequencing count-basedrare-variant detection assay for forensic analyses using highlycontrolled mixtures of synthetic plasmids and well characterizedBacillus anthraces genomic material. As long as the sequence of thetarget genomes and all the SNP loci are well understood, it isrelatively easy to identify regions with di-nucleotide or eventri-nucleotide haplotypes to target for minor variant sequence analysis.

Box 1. The Effect of Haplotype Analysis on Detection of Minor Variants

LOD=(Xr)(10)(100)

L=the lower limit (%) of minor variant detection

X=the average sequence error rate, and

n=the number of target SNP loci on a single sequencing read

In order to establish such an assay for Mtb, the Inventors adapted thehaplotype accuracy and sensitivity to Mtb resistance markers.Unfortunately, this haplotype approach is only applicable to SNP locithat can be detected on a single sequencing read. As most resistancemechanisms in Mtb are due to SNP loci in several different genomicallydispersed genes, it is not possible to generate a di-nucleotidehaplotype for every targeted resistance loci within a single sequencingread. To overcome this, the Inventors established a conceptually similarapproach of “Single Molecule with Overlapping Reads” (SMOR) analysis forindividual targeted SNP loci. With SMOR, the Inventors exploited themost recent advances in NGS technology by using longer over-lappingpaired end sequence reads to interrogate single loci twice in linkedsequencing reads. The Illumina MiSeq platform may be used with 300 bppaired end sequencing chemistry, which results in nearly completeoverlap of the forward and reverse reads at target loci. This allows foreffective coverage of each locus on both strands of an individualsequenced DNA molecule, which in turn allows for an independentconfirmation of the specific nucleotide at that single locus. A similarapproach of independently tagging each strand and sequencing separatelyhas been validated; however, the approach here is to sequence bothstrands consecutively. Again, the product rule of probability applies,such that if one locus on a single molecule is read two times, it hasthe same lower limit of detection as a haplotype of two distinct loci onone read. Therefore, if a particular SNP locus has a sequencing errorrate of 0.25% (typical for a high GC genome such as Mtb on Illuminatechnology), the limit of accurate detection may beLOD=(0.0025²)(10)(100)=0.006% with the present invention.

FIG. 2 shows the number of paired complimentary strand reads forresistance-conferring SNPs in the gyrA and inhA genes in experimentallyderived mixtures of “pure” susceptible and “pure” resistant Mtb genomesat approximately 999:1 (˜0.3%), respectively. An analysis was conductedon a number of resistance genes (rrs, katG, inhA, and gyrA), providingample evidence for the proof of principle for this assay. Sequencingerror is readily detected and is easily separated from “true calls”.

FIG. 3 is a visualization of actual paired overlapping reads aligned tothe gyrA sequence, from a 99:1 susceptible to resistant mixture (1%),showing the presence of resistance allele and susceptible allele states(shading denotes sequences from paired reads).

Heteroresistance Detection Assay (HDA) Optimization

The Inventors devised a strategy to identify single resistanceconferring mutations down to 0.1% or less using the Illumina MiSeqtabletop sequencer, by making use of SMOR analysis. This strategy may beused in an assay for 36 markers on six resistance-associated genes:katG, inhA promoter, rpoB, gyrA, rrs, eis promoter. Current MiSeqthroughput allows for up to 96 patient samples (with up to 6 targetgenes) in a single run and still obtain >10⁵ reads per target, allowingfor variant detection to less than 0.1% (LOD˜10⁻⁴), if present. Thefinal assay included a Mtb-specific quantitative PCR analysis of eachclinical sample to establish quantity of Mtb present in the sample.

HDA Standards

The assay may include a highly homogeneous synthetic plasmid DNAstandard that contains each well-described resistance conferringmutation, for up to 6 genes (˜36 SNPs). Deep sequencing of the standardenables identification of actual sequence error rate variance betweenloci and sequencing runs. The standards are used as error controls forthe HDA assays.

HDA Validation

A two-pronged approach is used to validate the HDA: 1) Applying the HDAto known mixtures of pan-susceptible and pan-resistant strains, toassess the assay's accuracy and level of detection for each target; 2)Validate its ability to accurately detect minor variant in clinicalsamples using normal flora sputum spiked with target DNA, to assessimpact of human and background microbial DNA on assay performance.

Example 2 Heteroresistance Analysis of Clinical Tuberculosis PatientSpecimens

The premise of this study was that Mtb heteroresistance can be detectedin patient samples prior to treatment failure. Dynamic changes can bequantified in Mtb populations and heterogeneity found in serial clinicalspecimens in order to track the population dynamics of pre-existing oremergent resistant sub-populations and to determine at what levelsub-populations of resistant organisms of the total Mtb populationpredict acquired drug resistance and poor treatment outcomes.

The goal was to quantify dynamic changes in Mtb heteroresistance toisoniazid (INH), rifampin (RIF), moxifloxacin (MOX), amikacin (AMK),kanamycin (KAN) and/or capreomycin (CAP) in a prospective study oftuberculosis (TB) and drug-resistant tuberculosis (DR-TB) patientsthrough the first nine months of their treatment or retreatment. TheInventors wanted to determine if the presence and/or expansion of minorvariant Mtb subpopulations with resistant alleles are associated withpoor clinical outcomes such as time to sputum and culture conversion andclinical improvement.

The project leverages the existing NIH-sponsored Global Consortium forDrug-resistant TB Diagnostics (GCDD) clinical study results andinfrastructure. The Inventors' group (GCDD) completed whole genomesequencing analysis of over 400 clinical multi/extensively drugresistant tuberculosis (M/XDR-TB) isolates from India, Moldova,Philippines and South Africa to quantifying the sensitivity andspecificity of individual SNPs and groups of SNPs as markers ofclinically relevant phenotypic resistance. Based on this work, theInventors determined that detection of 30-50 specific SNPs in six genes(katG, inhA promoter, rpoB, gyrA, rrs and eis promoter) detects 90-98%of Mtb isolates with clinically relevant phenotypic resistance to INH,RIF, FQ, AMK, KAN and CAP with almost 100% specificity. The GCDD globalinfrastructure has validated fluorescent smear microscopy, tuberculosisidentification, MGIT DST (1st and 2nd line drugs), and DNA extraction(used for line probe assays and pyrosequencing) at the PhthisisPneumology Institute Laboratory (Chisinau, Moldova), along with clinicalsites in Mumbai, India and Port Elizabeth, South Africa. The Inventorsenrolled 225 study participants in Moldova, 586 in India, and 275 inSouth Africa, all suspected of having MDR-TB, into a study focused onreducing the time of detection of XDR-TB from months to one week.

Heteroresistance Analysis

A sample analysis pipeline was established (FIG. 4). Briefly: (A) Samplecollection and extraction are accomplished with standard methods; (B)qPCR is run targeting the single copy rpoB gene, to establish a limit ofdetection for each sample based on the actual quantity of genomes persample; (C) Six gene regions of the Mtb genome (katG, inhA promoter,rpoB, gyrA, rrs, eis promoter; all of which contain the 42 differentresistance-conferring SNP loci, are amplified using hi-fidelitypolymerase,; (D) All six amplicons are pooled together for each patientat each time point in equal molar concentration; (E) A singlenext-generation sequencing Illumina library preparation is made for eachamplicon pool; (F) Several different NGS libraries (up to 96) are pooledtogether in equal molar concentration for sequencing on the MiSeq (aplasmid control is sequenced with every sequencing pool to examine errorof individual runs on the sequencing instrument); and (G) Abioinformatic analysis is conducted of all target loci, and the ratio ofnon-resistant to resistant allele states is quantified using in-housedeveloped and publically available scripts including SMOR analysis.

The contents of all references, patents, and published patentapplications cited throughout this application, as well as the Figures,are incorporated herein by reference in their entirety for all purposes.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

REFERENCES

So as to reduce the complexity and length of the Detailed Specification,the Inventors herein expressly incorporate by reference to the extentapplicable, all of the following materials.

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What is claimed is:
 1. A method of detecting a heteroresistantpopulation of a pathogen in a sample, the method comprising: a)providing a sample comprising a population of a pathogen; b) extractingnucleic acids from the sample; c) amplifying a target locus of thegenome of the pathogen in the extracted nucleic acids, wherein thetarget locus comprises at least one minor variant associated with drugresistance in the pathogen; d) consecutively sequencing both nucleicacid strands amplified from a single DNA molecule of the target locus ona Next Generation Sequencing (NGS) platform; e) applying an alignmentalgorithm to sequencing data from the overlapping nucleic acid strands;and f) performing an analysis of the aligned sequencing data to detectthe at least one minor variant and heteroresistant population of thepathogen.
 2. The method of claim 1, wherein the analysis of the alignedsequencing data is a minor variant analysis.
 3. The method of claim 2,wherein the minor variant analysis is a haplotype variant analysis. 4.The method according to any one of the preceding claims, wherein thelimit of detection for the minor variant is less than about 0.1% of theheteroresistant population.
 5. The method according to any one of thepreceding claims, wherein the target locus is amplified with a highfidelity polymerase.
 6. The method according to any one of the precedingclaims, wherein each of the overlapping nucleic acid strands consists ofless than about 300 nucleotides.
 7. The method according to any one ofthe preceding claims, wherein the alignment algorithm is optimized forshort nucleotide space reads of less than about 300 nucleotides.
 8. Themethod of claim 7, wherein the alignment algorithm is Novoalign.
 9. Themethod according to any one of the preceding claims, wherein the NGSplatform is an Ilumina MiSeq platform.
 10. The method according to anyone of the preceding claims, wherein the minor variant analysis isperformed with a bioinformatics script that requires a user to inputgenomic regions of interest and generates a report with singlemolecule-overlapping read information used to identify the minorvariant.
 11. The method according to any one of the preceding claims,further comprising: performing steps c) to f) of claim 1 with a highlyhomogenous synthetic plasmid standard to identify actual sequence errorrate variance between target loci and sequencing runs.
 12. The methodaccording to any one of the preceding claims, wherein the minor variantis selected from the group consisting of a single nucleotidepolymorphism (SNP), an insertion, and a deletion.
 13. The methodaccording to any one of the preceding claims, wherein the pathogen isMycobacterium tuberculosis.
 14. The method of claim 13, wherein the atleast one minor variant is located within a genomic sequence selectedfrom the group consisting of katG, inhA promoter, rpoB, gyrA, rrs, eispromoter, and combinations thereof.
 15. The method according to any oneof claims 13 to 14, wherein the heteroresistant population is resistantto isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), amikacin (AMK),kanamycin (KAN) and/or capreomycin (CAP).
 16. The method according toany one of claims 13 to 15, further comprising: administering atherapeutic agent to the heteroresistant population of M. tuberculosis,wherein the therapeutic agent is selected from the group consisting ofPA-824, OPC-67683, SQ109, TMC207, NAS-21, NAS-91, thioridazine,chlorpromazine, and a 1,3-benzothiazin-4-one, and combinations thereof.